Determination of cyclosporine concentration in the body is important for supervision of therapy in patients treated with drugs which contain the therapeutically significant stripstance group of cyclosporines.
Cyclosporine is it cyclic polypeptide immunosuppressant agent consisting of eleven amino acids. It is usually produced as metabolite by the fungus species Tolypocladium inflatum Gams. Chemically cyclosporine is [R-{R*, R*-(E)}]-cyclic-(L-alanyl-D-alanyl-N-methyl-L-leucyl-N-methyl-L-leucyl-N-m ethyl-L-valyl-3-hydroxy-N-,4-dimethyl-L-2-amino-6-octenoyl-L-.alpha.-amino- butyryl-N-methylglycyl-N-methyl-L-leucyl-L-valyl-N-methyl-L-leucyl). Cyclosporine generally prolongs survival of allogencic transplants involving skin, heart, kidney, pancreas, bone marrow, small intestines, and lungs. It generally also suppresses some humoral immunity and to a greater extent cell-medicated reaction such as allograft rejection, delayed hypersensitivity, experimental allergic encephalomyelitis, Freund's adjuvant arthritis and graft vs. host disease in many animal species for a variety of organs.
Cyclosporine is the primary choice for prophylaxis of organ rejection in kidney, liver, and heart allogeneic transplants. They are generally always used with adrenal corticosteroids. The drug is also used in the treatment of chronic rejection in patients previously treated with other immunosuppressant agents. The use of cyclosporine presents a number of risks, and, therefore, its bioassay and prevention of excessive bioavailability are significant. In addition to the risk of anaphylaxis, when high doses are used, cyclosporine can also cause hepatotoxicity and nephrotoxicity. Furthermore, serum creatinine and BUN levels can also be unduly elevated during cyclosporine therapy. The aforementioned side effects are most often responsive to dosage reduction. Generally, cyclosporine therapy may require frequent dosage adjustment.
In some cases poor absorption can result in difficultly in achieving required therapeutic levels.
For all of the foreclosing reasons, in addition to renal and liver functions assessment by measurement of BUN, serum creatinine, serum bilirubin, and liver enzyme, also the frequent bioassay of cyclosporine is of paramount importance.
It is to be understood that any reference throughout the specification and the claims to "cyclosporine", is intended to cover cyclosporine as well as its biologically active and pharmaceutically acceptable derivatives and which behave in a biological sense as cyclosporine.
Cyclosporine concentrations have to be determined in aqueous systems, primarily in biological liquids, particularly in blood serum or plasma, synovial liquid, urine, suspensions of blood cells, homogenized tissue. etc.
It is necessary, to measure the concentration of the bioactive material in the body liquids of patients to establish the usual relatively narrow therapeutically logical concentration range of the medication and to eliminate immunological reaction of transplant rejection in the case of unduly small dosage, and also to eliminate toxic side effects when the dosage is too high. Furthermore, the necessity of such concentration determinations is especially indicated by the individual differences between patients and the natural metabolic rate and rate of elimination of the active ingredients, and the individual changes of elimination rate in the case of medications over a longer period, all of which require a differentiation in the dosage of the drug.
The customary concentration assay of cyclosporine is carried out by means of immunological or chromatographic techniques known per se, such as from Keown et al.: "The Clinical Relevance of Cyclosporine Blood Levels as Measured by Radioimmunossay", Transplant Proc. (1983), 15, pp 2438-2441; and P. K Kabra, J. H. Wall, P. Dimson: "Automated Solid-Phase Extraction and Liquid Chromatography for Assay of Cyclosporine in Whole Blood". Clinical Chemistry (1987), 33, pp. 2272-2274.
The Keown et al. reference states that "Cyclosporine concentrations are measured both in serum, and in whole blood following erythrocyte lysis using the following two methods: (1) radioimmunoassay (RIA)--this was performed as described using the kit provided by Sandoz Limited Basel; (2) high-pressure liquid chromatography (HPLC)--this was performed using a Waters HPL chromatograph (Waters Associates, Bedford, Mass.), after acid and basic extraction to remove contaminating substances. The flow rate was 1.5 ml/min, and the CsA peak was read at 210 nm wavelength."
The Kabra article states that in this rapid, precise, accurate, cost-effective, automated liquid-chromatographic procedure for determining cyclosporine in whole blood, the cyclosprine is extracted from 0.5 mL of whole blood together with 300/.mu.g of cyclosporin D per liter, added as internal standard, by using an Advanced Automated Sample Processing unit. The on-line solid-phase extraction is performed on an octasilane sorbent cartridge, which is interfaced with a RP-8 guard column and an octyl analytical column, packed with 5-.mu.m packing material. Both columns are eluted with a mobile phase containing acetonitrile/methanol/water (53/20/27 by vol) at a flow rate of 1.5 mL/min and column temperature of 70.degree. C. Absolute recovery of cyclosprorine exceeded 85% and the standard curve was linear to 5000/.mu.g/L. Within-run and day-to-day CVs were &gt;8%. Correlation between automated and manual Bond-Elut extraction methods was excellent (r=0.987). None of 18 drugs and four steroids tested interfered."
The immunological assays are based on the production of appropriate antibodies against the cyclosporine in question. The concentration of the antigen can be determined with the aid of such antibodies and available immunoassay techniques. A major problem of this method is that it requires a great deal of manual operation and the possibility of crossover reactions with therapeutically inactive compounds.
Chromatographic methods employ the separation by means of physical effects of the compound to be analyzed, from substances which would affect the accuracy of the determination. Therefore, the sensitivity of the analysis is strongly affected by the quality of the detector and also by the quality of the separation. A special drawback of this method is that relatively large amounts of material are required to determine the cyclosporine concentration e.g. in blood plasma, as well as the requirement for special apparatus.